The present invention relates to an apparatus for investigating the kinetics of fast chemical reactions in solution using relaxation and/or mixing methods with spectrophotometric observation of the time course of reaction.
Various measuring techniques are known for these investigations. They are based on the direct observation of the equilibrium following an initial non-equilibration process state of a chemical system. This initial state can either be established by mixing several reactants (rapid mixing, especially stopped-flow techniques) or by external perturbation of a previously existing equilibrium state ("relaxation techniques").
The article Rev. Sci. Instr. 22, 619 (1951) describes a mixing technique in which the reactants are quickly mixed in a chamber containing a number of mixing jets. The mixture then flows into a "flow"-tube through which a light beam passes. The light intensity behind the flow-tube is a measure of the concentration of the reaction product, and is recorded as a function from the distance of the mixing chamber, which gives the elapsed time of reaction (continuous flow method). In another flow technique the reacting mixture is stopped within a few milliseconds by a movable piston which has a stop position. After the flow is stopped, the time course of reaction is measured by the absorption of visible or ultraviolet light, or other optical parameters (stopped-flow method: J. Physiology 117, 49 P (1952)).
In contrast to flow techniques, relaxation techniques start from the equilibrium state of the chemical system. A non-equilibrium state which deviates only by a small extent from the former equilibrium state is produced by a sudden change of temperature, pressure, or electric field strength. Use of these methods offers the advantages of a considerably smaller amount of substance, a higher time resolution and an easier mathematical analysis of the results. These methods are characterized as temperature-, pressure-, and field-jump techniques, depending on the kind of perturbation applied. The initial non-equilibrium state can also be achieved by electromagnetic irradiation, especially with high intensity light (flashlight photolysis). A general survey of the various techniques used for studying fast reactions will be found in Techniques of Chemistry, Vol. VI, Part II, ed. G. G. Hammes, Wiley-Interscience, 3rd edition (1974).
In case of the temperature-jump methods the sudden temperature increase is usually produced by discharging a high voltage capacitor. The discharge current passes through the electrically conducting test substance via a spark gap and two metal electrodes which are in electric contact with the test substance. Within a few microseconds or less it is possible to achieve temperature changes of several degrees centigrade. Another method used is to quickly heat the test substance by irradiating it with a microwave or a giant laser pulse. For measurements in the long-time range of 10secs or more, fast switching or liquid thermostatting of the sample cell has been used (Europ. J. Biochem. 4, 373 (1969)).
A review of the arrangements used for temperature-jump methods is given in the paper of A. Yapel and R. Lumry "A Practical Guide to the Temperature-Jump Method for Measuring the Rate of Fast Reactions", published in Methods of Biochemical Analysis, ed. D. Glick, Vol. 20, p. 169-350, Interscience N.Y. For studying intermediate, e.g. in enzyme reactions, a combination of the rapid mixing and the temperature-jump method can be used, e.g. as described in Rev. Sci. Inst. 37, 746 (1966).
Pressure-jump methods use an autoclave cell chamber or, for measurements in the .mu.sec-range, a shock-tube arrangement. The fast pressure change is usually produced by suddenly bursting a metal disk. (W. Knoche in Techniques of Chemistry, loc. cit.)
In the case of electric field-jump methods the non-equilibrium state is produced by suddenly applying a high electric field. This results in a dissociation of weak electrolytes. (L. De Maeyer in Methods in Enzymology, vol. 16, eds. Kustin, Colowick, and Kaplan, Academic Press (1969); L. De Maeyer and A. Persoons in Techniques of Chemistry, loc. cit.)
Optical methods are usually used for observing temperature-jump and field-jump techniques. For pressure-jump techniques conductivity changes are mostly measured, but optical absorption can also be used Analyt. Biochemistry 28, 273 (1969).)
For producing a non-equilibrium state by radiant energy the side of a transparent reaction vessel is exposed to radiation from the short, powerful gas discharge of a flashlamp. A continuous or pulsed probing light beam passes the axis of the vessel. A pulse laser can also be used instead of the flashlamp. A review of photochemical flashlight devices is given by G. Porter and M. A. West in Techniques of Chemistry, loc. cit.
For a review of apparatus with special reference to commercial set-ups see also: Z. A. Schelly and E. M. Eyring in Journal of Chemical Education, 48, A 639 and A 695 (1971).
The existing techniques are based on the combination of a specific perturbation method with a specific observation method. Consequently, measurements using the temperature-jump, pressure-jump, field-jump, flashlight- and flow-techniques always required a specially designed apparatus. Changing the parameter of observation (absorption, fluorescence of polarimetric methods) involved at least troublesome modifications and readjustments of the apparatus. Performance of experiments with various physical parameters of perturbation and observation was therefore largely restricted, especially comparative measurements using various parameters.
The need for designing special apparatus was very common in order to obtain maximal sensitivity (which is generally required in research problems). To illustrate this point apparatus for combined flow-temperature jump measurements should be mentioned. If such an apparatus is derived from a satisfactory flow apparatus satisfactory temperature-jump measurements cannot be obtained, and vice versa. On the one hand, the concentration changes involved in temperature-jump measurements are considerably smaller than those in flow-measurements. They are often less than 1% and their time course has to be determined quantitatively in periods which are up to 1000 times shorter. As a consequence, the signal-to-noise-ratio should improve but it deteriorates at short time periods because of the too low light intensity. On the other hand, highly spcialized temperature-jump apparatus are very sensitive to mechanical vibrations which occur in flow experiments. Therefore, they show troublesome instabilities of the light flux if used for combined flow-temperature jump measurements. An apparatus optimized for the combined measurements, however, does never give the same sensitivity as a pure temperature-jump or a pure stopped-flow apparatus when used for uncombined measurements. Thus the number of apparatus needed for optimized work is still increased.